Project Summary: There is growing evidence that an overexuberant innate and adaptive immune response may contribute to life-threatening pulmonary pathology in COVID-19 disease. On the other hand, inadequate viral control may allow severe disease to develop. Human immune system (HIS) mouse models have enormous and unique potential to model human COVID-19. Unlike other animal models, HIS mice could be used to understand the role of the innate and adaptive human immune systems in both controlling and driving SARS- CoV2-mediated disease and hence be used to optimize therapeutic approaches. The goal of our proposal is to optimize HIS mouse models for these purposes. Specifically, we propose to: 1) Optimize our HIS mouse models for the study of COVID-19. Existing mouse models are limited by the lack of human ACE2, the SARS- CoV2 receptor, in the respiratory tract. We will implant iPS cell-derived human lung bud organoids generated from cord blood HSC donor cells into HLA-A2 Tg NSG mice receiving HLA-A2+ cord blood HSCs. In a second approach to humanizing mice for COVID-19 mouse studies, we will use CRISPR/Cas9 to replace the murine ACE2 gene with hACE2, allowing physiologic expression of hACE2 in NSG mice. Human HSC recipients will be treated with mouse TSLP in an AAV vector to enhance murine thymic and lymph node structure and thereby improve human T cell development and improve peripheral vaccination responses. An alternative approach to enhancing thymus function will involve grafting of multiple pieces of thymocyte-depleted neonatal human thymus tissue in multiple sites to compensate for the lack of growth potential (compared to fetal thymus) of neonatal thymus tissue. Immune reconstitution, T cell reconstitution and lymphoid structure will be followed and humoral and cellular responses to live attenuated SARS-CoV2 virus vaccination will be measured; 2) Use optimized HIS mouse models to determine the kinetics of disease pathogenesis and the role of human immune components in controlling infection and mediating pathologic host responses. We will first employ HIS mice constructed with human cord blood HSCs and autologous iPSC-derived lung bud implants and later utilize the above HLA-A2 hACE2 Tg model. Baseline infection with SARS-CoV2 will be assessed in non-reconstituted animals and compared to HIS mice. In HIS mice, we will deplete various human immune components (T cells, B cells or macrophages) to determine their impact on the course of infection and pathology associated with SARS-CoV2. In hACE2 Tg mice we will investigate the kinetics of infection of various components of the respiratory tract in combination with analysis of the human immune cell infiltrates in each locale over time. With these models established, we will be positioned to test therapeutic approaches during different phases of SARS-CoV2 infection in future studies. Collectively, our models will provide critical information on the role of human immune components in driving and protecting from COVID-19-associated pathology, allowing accelerated optimization of immunomodulatory therapies.